1. Field of the Invention
The present invention relates to a method for producing nitrile hydratase (NHase) utilizing a host of bacterium not belonging to the genus Rhodococcus.
2. Description of Related Art
Nitrile hydratase (NHase) is an enzyme that is produced in microorganisms and converts a nitrile compound into an amide compound by hydration. It is a soluble metalloprotein containing iron or cobalt atom in its active center. NHases have been isolated from several kinds of bacterial cells, and all of them consist of two kinds of subunits, .alpha. and .beta.. Both of the subunits have a molecular weight of about 23,000. It has also been reported that NHase derived from Rhodococcus sp. N-771 strain has a non-heme iron center of mononuclear low-spin six coordinate Fe(III). NHases of Rhodococcus sp. N-771, N-774 and R312 are considered to be the same enzyme because their nucleotide sequences are identical to one another, and their enzymatic activity varies with light irradiation. That is, when bacterial cells exhibiting high activity are left in the dark, the enzyme activity is reduced, and the activity is increased again by photo-irradiation. This is because, in the dark, nitric oxide produced from the cell bonds to the active center of the iron type nitrile hydratase, i.e., the non-heme iron center, to produce an inactive state. When the iron type nitrile hydratase in this inactive state is irradiated by light, NO is instantly dissociated, and the iron type nitrile hydratase is activated. The above binding characteristic of NO is commonly observed for substantially all of the iron type nitrile hydratases.
The present inventors are attempting to apply the above photoreactive iron type nitrile hydratase derived from Rhodococcus bacteria to, for example, increasing efficiency of amide production, photoreactive amide production and the like. To this end, however, it is necessary to improve the iron type nitrile hydratase derived form Rhodococcus bacteria, for example, to further enhance its catalytic ability. To improve the catalytic ability, it can be expected to use genetic recombination techniques utilizing a widely usable host such as Escherichia coli. In fact, the nitrile hydratase gene has been isolated, and the production of the enzyme by utilizing Escherichia coli has also been reported. However, according to the previous reports, when Escherichia coli was used as a host, the specific activity per unit weight was lower than that obtained by utilizing a Rhodococcus bacterium as the host. Therefore, Rhodococcus host vector systems have conventionally been used in general. However, handling of Rhodococcus bacteria is more difficult compared with Escherichia coli and the like. In addition, since any potential promoter therefor is not available, they suffer from problems, for example, sufficient increase of activity can not be obtained.
As mentioned above, various reports have previously been made for the recombinant production of nitrile hydratase utilzing Escherichia coli. According to those reports, it has been elucidated that, in both of the iron type and cobalt type nitrile hydratases, it is required for their expression to co-express another gene which is present in the nitrile hydratase operon in addition to the genes for the .alpha. and .beta. subunits constituting nitrile hydratase. As for the cobalt type nitrile hydratase, the nitrile hydratase has been successfully produced by simultaneously introducing these three kinds of genes into Escherichia coli. A similar approach was expected to be effective as also for the iron type enzyme, and the nitrile hydratase from a Pseudomonas bacterium has successfully produced in Escherichia coli.
However, the aforementioned methods cannot be considered to be an efficient method. In addition, when the photoreactive iron type nitrile hydratase derived form Rhodococcus bacteria is produced by using Escherichia coli as the host, most of the produced nitrile hydratase precipitates, and therefore the activity of the recombinant cell becomes extremely low. Therefore, it is difficult to practically utilize the produced nitrile hydratase in such a method, which has been a serious obstacle for utilization of the photoreactivity.
However, if it becomes possible to efficiently produce the photoreactive iron type nitrile hydratase derived from Rhodococcus by using Escherichia coli, it will enable to enhance the catalytic ability, and make it easy to utilize the enzyme for increasing amide production efficiency and the photoreactive amide production.